The invention relates to methods of identifying compounds useful in the field of flavours and fragrances, and to the corresponding lead structures of these compounds. Said methods comprise a reaction with a metabolic enzyme. The invention also relates to the identification of metabolic enzymes and their use in said methods.
Compounds useful in the field of flavours and fragrances may be flavour compounds and fragrance compounds as such, but also modulators of fragrance and flavour perception. Such modulators include enhancers and masking agents of the olfactory and gustatory senses, and regulators or modulators of metabolic reactions involving aforementioned compounds occurring in the respiratory tract, in particular in the oral and/or nasal cavity.
Flavour and fragrance compounds reach the oral and nasal cavity where they may cause the perception of flavour and fragrance compounds by binding to olfactory and/or gustatory receptors. Binding to a receptor will lead to perception if the receptor is activated and initiates a signal transduction cascade that, if successfully transmitted, leads to the flavour or fragrance signal being perceived.
For olfactory receptors, almost 1000 genes have been identified on human chromosomes and it is speculated that approximately 350 different functional receptor proteins are present in the human nose to detect a plethora of odorous compounds. Typically, an odorant reaches the olfactory epithelium to bind and activate olfactory receptors.
The use of olfactory and gustatory receptors in in vitro chemoreceptor screening methods to identify new flavour and fragrance compounds and lead structures is known and described for example in WO9217585, WO02059349, US20020064817, WO0127158. In these screening methods, chemoreceptors are exposed to compounds and identify receptor ligands based on the interaction of a compound with an olfactory or gustatory receptor of interest.
However, known screening methods identify compounds or leads that in many cases turn out to be not relevant.
It has been speculated that compounds may undergo enzymatic metabolism in the respiratory tract and in the oral and nasal cavity, and form derivatives with altered chemical, physical and biological properties. Enzymatic metabolism has not yet been shown in the human olfactory epithelium in vivo, and only a small number of genes encoding metabolic enzymes have been previously reported to be expressed in the human nose or oral cavity (carboxyl esterase enzymes, a UDP-glucuronosyltransferase UGT2A1, and cytochrome P450 enzyme CYP2A13).
Receptor ligands identified by known in vitro receptor screens are not necessarily perceived as fragrance or flavour. Whether a ligand actually triggers fragrance or flavour perception in the human nose depends on the fate of the ligand which may depend on the occurrence of metabolism. By the action of a metabolic enzyme, the ligand may react to a compound that will not bind any receptors and is not perceived by the human senses at all. Furthermore, another receptor ligand may be generated to account for a different quality of perception, which makes it very difficult to find lead structures.
Metabolism of such compounds that are substrate of an enzyme present in the oral or nasal cavity or the respiratory tract may occur after receptor activation, or may occur prior to receptor binding in the fluidic mucus or in cells lining the cavity. The metabolite(s) may have chemical and/or physical properties which are of advantage for interaction with receptors, other enzymes and/or odorant binding proteins. Substrates may be odorant compounds or non-odorant compounds. In case of the latter, one or more metabolite of said substrate may be an odorant, and/or have the above-mentioned properties.
Metabolism may inactivate or activate receptor ligands. Fragrance and flavour compounds may be agonists, antagonists, enzyme substrates, enzyme inhibitors, and allosteric regulators of receptors or enzymes. The metabolites may compete for example for receptor binding, interact with additional receptors and enzymes, and/or modulate the activity and sensitivity of receptors and enzymes and components of the signal transduction cascade, including cyclic nucleotide-gated (CNG) channels of olfactory sensory neurons. The metabolites generated from substrates of metabolic enzymes may have properties which enable them to interact with receptors and enzymes and these metabolites may in fact be primarily responsible for the perceived quality and effects of flavour and fragrance ingredients and/or compete with their substrates for receptor interaction, and in particular for receptor activation.
Depending on the occurrence of enzymatic metabolism in the oral or nasal cavity or the respiratory tract, known in vitro chemoreceptor screening methods may result in false positive or false negative results when trying to identify fragrances or flavours. For example, a ligand identified by the screening method may be rapidly metabolised in the human nose to a non-olfactive compound (false positive result). Another problem occurs if a compound that is not a ligand to a chemoreceptor itself but is metabolised in the human nose to result in a ligand for a chemoreceptor, i.e. a precursor. The precursor will give a negative signal when applying known screening methods (false negative result). These possibilities are further illustrated below for an olfactive compound “A”.
Compound “A” has a particular olfactive note described by a perfumer. A is metabolised to compound “B” in the nose and B is responsible for the particular note, by activating one or several olfactory receptors which are required to perceive the smell as described by the perfumer. In an in vitro receptor screen performed with A (precursor), the receptors responding to B are not activated and will not be identified. The screen will either give a correct negative result for A while failing to identify receptors responding to B (“failed identification”), or identify receptors which respond to A but are not relevant since A is metabolised to B (“false positive results”). Known screens show the correct structure-activity relationship. However, the identified compound may still be irrelevant regarding the perception as fragrance, so that the screen will not indicate a correct relationship between structure and fragrance perception. This will make subsequent identification, for example on the basis of lead structures, difficult or impossible.
Even if a compound is correctly identified by a conventional receptor screen it may be very difficult to find the relevant lead compounds on the basis of these results, since the lead may be incorrect and misleading. The example below illustrates this.
A compound “C” may be partially metabolised in the human nose to form compound “D”, both compounds being present in the nose in parallel. “C” and “D” may have different olfactive notes, which may account for the broad olfactive description assigned to some single fragrance or flavour compounds. Known screening methods to identify flavours and fragrances make lead finding for particular olfactive notes very difficult, since the identified ligand may not be responsible for the fragrance or flavour perception in the human nose at all. Therefore lead compounds identified for particular olfactive notes may not be the relevant ones.